Modrzejewski et al. Environ Evid (2019) 8:27 https://doi.org/10.1186/s13750-019-0171-5 Environmental Evidence

SYSTEMATIC MAP Open Access What is the available evidence for the range of applications of genome‑editing as a new tool for plant trait modifcation and the potential occurrence of associated of‑target efects: a systematic map Dominik Modrzejewski* , Frank Hartung, Thorben Sprink, Dörthe Krause, Christian Kohl and Ralf Wilhelm

Abstract Background: Within the last decades, genome-editing techniques such as CRISPR/Cas, TALENs, Zinc-Finger Nucle- ases, Meganucleases, Oligonucleotide-Directed Mutagenesis and base editing have been developed enabling a precise modifcation of DNA sequences. Such techniques provide options for simple, time-saving and cost-efective applications compared to other breeding techniques and hence genome editing has already been promoted for a wide range of plant species. Although the application of genome-editing induces less unintended modifcations (of-targets) in the genome compared to classical mutagenesis techniques, of-target efects are a prominent point of criticism as they are supposed to cause unintended efects, e.g. genomic instability or cell death. To address these aspects, this map aims to answer the following question: What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modifcation and the potential occurrence of associated of-target efects? This primary question will be considered by two secondary questions: One aims to overview the market-ori- ented traits being modifed by genome-editing in plants and the other explores the occurrence of of-target efects. Methods: A literature search in nine bibliographic databases, Google Scholar, and 47 web pages of companies and governmental agencies was conducted using predefned and tested search strings in English language. Articles were screened on title/abstract and full text level for relevance based on pre-defned inclusion criteria. The relevant infor- mation of included studies were mapped using a pre-defned data extraction strategy. Besides a descriptive summary of the relevant literature, a spreadsheet containing all extracted data is provided. Results: Altogether, 555 relevant articles from journals, company web pages and web pages of governmental agen- cies were identifed containing 1328 studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Most of the studies were conducted in China followed by the USA. Genome- editing was already applied in 68 diferent plants. Although most of the studies were basic research, 99 diferent mar- ket-oriented applications were identifed in 28 diferent crops leading to plants with improved food and feed quality, agronomic value like growth characteristics or increased yield, tolerance to biotic and abiotic stress, herbicide toler- ance or industrial benefts. 252 studies explored of-target efects. Most of the studies were conducted using CRISPR/ Cas. Several studies frstly investigated whether sites in the genome show similarity to the target sequence and secondly analyzed these potential of-target sites by sequencing. In around 3% of the analyzed potential of-target

*Correspondence: dominik.modrzejewski@julius‑kuehn.de Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Erwin‑Baur‑Straße 27, 06484 Quedlinburg, Germany

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Modrzejewski et al. Environ Evid (2019) 8:27 Page 2 of 33

sites, unintended mutations were detected. Only a few studies conducted of-target analyses using unbiased detec- tion methods (e.g. whole genome sequencing). No of-target efects that could be correlated to the genome-editing process were identifed in these studies. Conclusions: The rapid adoption in plant breeding was demonstrated by a considerable number of market oriented applications (crops and traits) described in publications worldwide. Studies investigating of-target efects are very heterogeneous in their structure and design. Therefore, an in-depth assessment regarding their weight of evidence is mandatory. Keywords: New plant breeding techniques, Gene editing, Targeted genome modifcation, Mutagenesis, Unintended efects, Of-target mutation, Evidence map, Evidence synthesis

Background meaning that yield improvement conserves the rate of Technological progress in agriculture and plant breeding land clearance, but the efect is smaller than it could be has contributed signifcantly to a stable food supply and [5]. One example for the rebound-efect in agriculture is formed the basis for high yields as well as the production the Green Revolution [6]. As a result of this revolution, of high-quality agricultural products [1]. However, in an yields increased, saving ecosystems from conversion to ever-changing world, new challenges will be encountered agricultural land. However, the efect was much smaller within the next decades. Aside the demands of the grow- than expected. One explanation for this efect may be ing global population and limited fossil resources, climate that increased productivity due to new technologies also change is a driver of breeding eforts as it is associated increases the proftability of agriculture compared with with increased extreme weather events like droughts or alternative land use [6]. foods as well as changing dynamics of pests and dis- Plant breeding essentially relies on the utilization of eases. Agriculture needs to ensure and increase the world genetic variation within the breeding material that can agricultural production to serve extended demands with be used for crossing and selection to develop improved limited environmental resources like soil and water [2]. varieties. New genetic variation occurs naturally by spon- In contrast, intensifcation of agriculture causes consid- taneous mutations that enable some individual plants in erable impact on nature, as naturally diverse landscapes a population to adapt to changing environmental con- are replaced by arable land for the cultivation of few plant ditions. However, as the mutation rate is fairly low and species. Biodiversity is threatened through habitat loss random, plant breeders and scientists have artifcially and pesticide use, which in turn is considered to increase induced mutations for several decades [7]. Te frst gen- disease and pest pressure [1]. Additionally, these impacts eration of mutation breeding used chemical and physical of agriculture on the environment are becoming increas- mutagens to generate a plurality of nonspecifc muta- ingly important in societal debates. tions. Te increased mutation rate results in plants with To meet all these challenges, improved crop varie- a few positive, a lot of neutral and several negative char- ties may be developed and integrated into a sustainable acteristics. Tus, laborious backcrossing and selection farming system considering their economic, environ- steps are necessary in order to select for a target trait in mental and social impacts [2]. Examples for a sustain- a desired genetic background (maternal variety). Never- able farming system are the preservation of natural soil theless, to date 3282 mutant varieties from 225 diferent fertility through suitable crop rotations, fertilization plant species have been generated through undirected and plant protection according to the principles of inte- mutagenesis and ofcially but voluntarily registered [8]. grated cultivation or a balanced and species-appropriate animal husbandry [3]. Additionally, plant breeding is of Glossary: [1, 9, 10] crucial importance to manage environmental impacts Backcrossing on cultivation systems by providing varieties resistant to plant diseases or pests, tolerant to abiotic stress and Backcrossing is a crossing of a hybrid with one of its more broadly support a “greener production”—in time. parents in order to achieve ofspring that are geneti- Tis may reduce pesticide use and result in less intense cally closer to the selected parent. Tis way, desired management eforts (e.g. irrigation) [4]. Further yield heterologous traits from the hybrid can be transferred improvement can increase the yield per hectare and may into the genetic background of a parental line. Since open land management options e.g. balance with areas crossing recombines all genes, many backcrosses for nature conservation [1]. Nevertheless, there is evi- are necessary to achieve considerable dilution of dence that increased yields may lead to a rebound-efect unwanted genes from the hybrid. Modrzejewski et al. Environ Evid (2019) 8:27 Page 3 of 33

Mutagen 1. No template is added and the DSB is repaired by autochthonous cellular mechanisms [in most cases A mutagen is an agent that increases the mutation non-homologous end joining (NHEJ)] resulting rate within an organism or cell, e.g. X-rays, gamma- in small insertion-deletion (indel) mutations. Tis rays or chemicals like ethyl methane sulfonate approach is defned as SDN1. (EMS). 2. A repair template is added which, except for a few Mutation breeding nucleotides, is identical to the sequences in which the DSB is introduced. Ten, the DSB is repaired via A plant breeding approach using mutagens to enhance homology-directed repair (HDR), causing nucleo- genetic variation. Te resulting random mutations can tide substitution or, depending on the template used, generate new gene variations with positive traits that targeted indels of a specifc size. Tis approach is can be selected for further breeding. However, several defned as SDN2. of these mutations are negative and diminish the via- 3. Te repair template harbors a recombinant DNA bility of the plant. sequence additional to the homologous sequences in Of‑target efect due to genome editing which the DSB is made and the break is repaired via HDR, resulting in more complex alterations, i.e. the Unintended cleavage and mutations at untargeted insertion of foreign genes. Tis approach is defned as genomic sites with similar but not identical sequences SDN3. compared to the target site.

Selection Te Oligonucleotide-Directed Mutagenesis technique does not induce a DSB. Instead the introduced oligonu- A process in breeding by which the breeder chooses cleotide binds to the targeted DNA sequences and this only those individuals that show desired trait(s). sequence is then modifed by the cellular mechanism of mismatch repair [10]. Base editing is a recently developed approach enabling a targeted switch of DNA bases in a given frame into another without any DSB [17]. Genome-editing ofers substantial advantages com- In 1983, the frst recombinant DNA was delivered to pared to previous mutation breeding techniques and plant cells using Agrobacterium tumefaciens [11, 12]. conventional genetic engineering in terms of speed From this time on, it is possible to work at a single gene and precision. It provides the opportunity to selectively level with genetic material from any organism, generat- mutate or modify one or a few genes (SDN1, SDN2). In ing plants that cannot be bred conventionally. Neverthe- addition, it is now possible to precisely modify or selec- less, the induced mutations using chemical and physical tively replace (SDN3) entire genes from both closely as mutagens as well as the “classical” transgenic approach well as distantly related organisms [10]. By the use of show limited efciencies and unintended side efects due conventional genetic engineering, traces of recombinant to the random targeting [13]. DNA, from the used gene shuttle (e.g. bacteria, virus or In recent years, genome-editing techniques have been plasmid), persist in the modifed organism leading to developed enabling a more precise modifcation of clearly characterized genetically modifed organisms. In DNA sequences in a site-directed manner [10]. To date, contrast, by applying genome-editing, it is possible to genome-editing comprises three molecular approaches modify crops without inserting foreign DNA sequences that efciently induce targeted alterations in genomes: at all [18]. Terefore, some countries like USA, Canada, (i) Site-directed nucleases (SDN), including Meganucle- Brasilia, Argentina and others reduced the regulatory ases (MN), Zinc-Finger Nucleases (ZFN), Transcription burden for plant breeders [19]. Due to the simplicity, Activator-Like Efector Nucleases (TALENs) and Clus- time-saving and cost-efective application of genome- tered Regularly Interspaced Short Palindromic Repeats/ editing, it has already been applied in a wide range of cul- CRISPR associated protein (CRISPR/Cas), (ii) Oligonu- tivars. Genome-editing has been used for: cleotide-directed mutagenesis (ODM) and (iii) base edit- ing (BE). A detailed description of the single techniques i. Analyzing gene functions (e.g. efect of the RAV2 is summarized in Additional fle 1. Site-directed nucle- gene for salt stress in rice [20]). ases induce double-strand breaks (DSBs) in the DNA ii. Improvement of product quality (e.g. improved oil which are subsequently repaired by the autochthonous quality in soybean [21]). cellular mechanisms. Te type of repair can be catego- iii. Development of disease resistant varieties (e.g. rized in three main types [10, 14–16]: virus resistant cucumber [22]). Modrzejewski et al. Environ Evid (2019) 8:27 Page 4 of 33

iv. Developing of herbicide tolerant varieties (e.g. resist- the available evidence for the potential occurrence of ance to the herbicide chlorsulfuron in canola [23]). associated of-target efects. Furthermore, risk assessors v. Improved adaption to abiotic stress, (e.g. drought and decision makers are depending on the provision of tolerance in maize [24]). a reliable body of evidence to support conclusions about potential risks being associated with the application of Even in plants like hexaploid wheat, that were so far genome-editing. Tus, an overview of the available evi- largely inaccessible for targeted genetic alterations, the dence on the occurrence of of-target efects could be of simultaneous mutation of all six alleles was successfully crucial importance. performed [25]. Tese successful applications are open- ing up new dimensions for the scientifc plant breeding and agricultural community. Stakeholder engagement Compared to randomly induced mutations by chemi- Te systematic map question, the secondary questions cals or irradiation, the number of unintended mutations and the scope was designed by the review team refecting (of-target efects) is broadly reduced by genome-editing discussions with policy makers, authorities, regulators techniques [10]. Nevertheless, their application does not and academia requesting a broad overview on the avail- completely or per se exclude the occurrence of of-tar- able evidence about the application of genome editing in get efects. Of-target efects caused by genome editing plants and the potential occurrence of of-target efects. most likely occur in DNA sequences that are similar (not Troughout the review process there was no stakeholder identical) to the targeted one but are located at another engagement. As indicated in the systematic map proto- site in the genome. Mainly, they occur due to the lack of col the results of the map were discussed on a confer- exclusiveness and/or length of the recognition site [10]. ence with diferent stakeholders from this feld, including When analyzing of-target efects, one distinguishes besides others plant breeders, federal authorities, aca- between biased and unbiased detection methods [26]. To demia, farmer organizations and processing industry. date, the predominant approach for identifying of-target Stakeholder remarks are taken into account when prepar- efects is the biased approach consisting of two steps: (i) ing a systematic review based on the results of this map. Using sequence alignment programs, sites in the genome with high similarity to the target sequence are identifed, which are designated as potential of-target sites. Several Objectives of the map diferent tools like BLAST [27], Cas-OFFinder [28] or As genome-editing techniques are a promising tool to CRISPR-P [29, 30] are used to identify these potential of- revolutionize plant breeding, they are of particular rele- target sites. (ii) Te identifed individual DNA sequences vance to scientists, breeders, farmers but also to decision (potential of-target sites) are then analyzed for unde- and policy makers with regards to the broader agricul- sired mutations (of-target efects) using various detec- tural management and future challenges. Terefore, tion methods like mismatch-sensitivity endonuclease we wanted to provide a comprehensive and transparent assays, Sanger sequencing or targeted deep sequencing overview of the available evidence base concerning the [31]. All detection methods have their specifc advantages efects of genome-editing in plants. Te main objectives and disadvantages which are addressed in several reviews were: (e.g. [26, 32]). In contrast to the biased detection meth- ods, unbiased ones are used to identify of-target efects • Overview of the traits modifed by genome-editing in in a completely unrestricted way. Terefore, it requires model plants as well as in crops produced for agricul- genome-wide sequencing to identify of-target muta- tural production. tions anywhere in the genome and de novo defne of- • Overview of the available evidence about the occur- target sites [26, 32]. Depending on the detection method rence of of-target efects due to the use of genome- being used, the results of identifying of-target muta- editing techniques in model plants as well as in crops tions vary widely. Although genome-editing techniques produced for agricultural production. induce much less of-target efects compared to classical • Identifcation of the geographical distribution of mutagenesis techniques, these are an important point of genome-editing activities in plants worldwide. criticism as they may possibly cause genomic instability, • Identifcation of the volume of the available litera- cytotoxicity and cell death [33–35]. ture, evidence clusters and key characteristics of the Tis systematic map facilitates an objective debate evidence base to inform interested stakeholder com- by informing interested stakeholder communities in a munities. transparent and retraceable manner about the status of • Identifcation of knowledge gaps concerning the research, the progress of genome-editing in plants and occurrence of of-target efects in order to inform Modrzejewski et al. Environ Evid (2019) 8:27 Page 5 of 33

decision makers which future research might be Regularly Interspaced Short Palindro- needed for a risk assessment. mic Repeats/CRISPR associated protein • Assessment whether a specifc section of the avail- (CRISPR/Cas), Transcription Activator- able evidence base is suitable for an in-depth analyses Like Efector Nucleases (TALENs), Meg- by a systematic review. anucleases (MN), Zinc-Finger Nucle- ases (ZFN), Oligonucleotide-Directed Te primary question of the systematic map was: Mutagenesis (ODM), base editing (BE). “What is the available evidence for the range of appli- Outcome: Te alteration of the genome (i.e. inser- cations of genome-editing as a new tool for plant trait tion, deletion or substitution of nucleo- modifcation and the potential occurrence of associated tides) induced by the use of a genome- of-target efects”? editing technique. Additionally, the To answer this primary question, it was subdivided into occurrence of of-target efects was two secondary questions related to (1) the traits modifed assessed. by genome-editing and (2) the occurrence of of-target efects due to the use of genome-editing.

Secondary question one Methods Te methods used to conduct this systematic map were “What are the traits modifed by genome-editing in based on the Collaboration for Environmental Evidence model plants as well as in crops produced for agri- (CEE) systematic review guidelines [36]. Detailed infor- cultural production?” mation about the methods used to perform this system- atic map are presented in the published protocol [37]. A Population: Any model plant or crop produced for brief summary of these methods is provided here. agricultural production. Intervention: One of the following genome-editing Search for articles techniques was used to induce an altera- Te search string was composed of two parts: Te frst tion in the plant genome: Clustered part defned the population of interest comprising less Regularly Interspaced Short Palindro- specifc terms like crop, plant or seed as well as spe- mic Repeats/CRISPR associated protein cifc model plants and crops including their English and (CRISPR/Cas), Transcription Activator- Latin names. Te second part defned the intervention, Like Efector Nucleases (TALENs), Meg- i.e. the genome-editing technique applied to induce an anucleases (MN), Zinc-Finger Nucle- alteration in the plant genome (CRISPR, TALENs, ZFN, MN, ODM or BE). To test the comprehensiveness of the ases (ZFN), Oligonucleotide-Directed search strategy a scoping search was carried out and the Mutagenesis (ODM), base editing (BE). identifed records where tested against an a priori defned Outcome: Te alteration of the genome (i.e. inser- test library with articles of known relevance. Details on tion, deletion or substitution of nucleo- search settings and subscriptions can be found in Addi- tides) induced by the use of a genome- tional fle 2. Te following bibliographic databases were editing technique. searched whereby the search string was adapted to the specifc needs of each database to which it was applied to: Secondary question two • Scopus “What is the available evidence for the potential • PubMed occurrence of associated of-target efects due to the • Science direct use of genome-editing in model plants as well as in • Agris crops produced for agricultural production?” • Web of Science (WoS) • Biological Abstracts • BIOSIS Previews Population: Any model plant or crop produced for • CAB Abstracts agricultural production. • SciELO Citation Index Intervention: One of the following genome-editing techniques was used to induce an altera- In addition, Google Scholar (https​://schol​ar.googl​ tion in the plant genome: Clustered e.com) was searched using 30 diferent combinations Modrzejewski et al. Environ Evid (2019) 8:27 Page 6 of 33

of diferent (model) plants and genome-editing terms. the plant genome: Clus- Te frst 20 hits organized by relevance, of each search tered Regularly Inter- term were examined for relevance. Furthermore, a total spaced Short Palindromic amount of 47 web pages of companies working with Repeats/CRISPR associ- genome-editing and the USDA database “Am I regu- ated protein (CRISPR/ lated?” (https​://www.aphis​.usda.gov/aphis​/ourfo​cus/ Cas), Transcription Acti- biote​chnol​ogy/am-i-regul​ated) were searched to identify vator-like Efector Nucle- grey literature. Finally, the bibliographies of 107 review ase (TALENs), Zinc- articles identifed by the literature search were screened Finger Nuclease (ZFN), to identify further relevant papers. In May 2018, the Meganuclease (MN), search string was applied in order to identify articles pub- Oligonucleotide-Directed lished after 1996, when the frst study about a genome- Mutagenesis (ODM), editing technique was published. base editing (BE). Eligible outcome for Article screening and study eligibility criteria secondary question one: An alteration in the plant Screening process genome was reported Before applying the selection criteria at title/abstract and (insertion, deletion or then at full text level, a consistency check was conducted substitution) due to the by all four participating reviewers aiming to determine use of a genome-editing the inter-reviewer agreement. Te level of agreement was technique. tested formally using a kappa test [38]. 100 references Eligible outcome retrieved by the search were randomly explored at title/ for secondary question two: Te occurrence of of-tar- abstract level leading to a kappa value of 0.48. After dis- get efects was assessed. cussing all disagreements, a second consistency check Eligible type of data: Only those references was carried out using another randomly allocated 100 were included which references resulting in a kappa value of 0.71. After title/ comprise primary data abstract screening, potentially relevant articles were checked at full text level. A list of unobtainable articles referring to the use (Additional fle 3) and articles excluded at full text level of a genome-editing with the reason for exclusion (Additional fle 4) are pro- technique to induce a vided. For conducting the consistency check as well as for sequence alteration in the the screening process at title/abstract and full text level plant genome. the open-access and non-proft database CADIMA was Eligible languages: References in German used [39]. Two members of the review team are authors and English languages of a few articles retrieved by the review process. How- were included. Articles ever, as none of these papers comprise primary data, in other languages were their articles were excluded at title/abstract level. Never- included when besides theless, the two coauthors routinely screened literature title and abstract, further covering the issues addressed in this map. parts of the article, like fgures or tables, were in Eligibility criteria English or German and An article had to meet all the following inclusion criteria the provided informa- in order to enter into the systematic map: tion allowed for a def- nite judgment of their Eligible populations: Any model plant or crop relevance. produced for agricultural production as well as Study validity assessment higher fungi was used. Eligible interventions: At least one of the fol- Te aim of this systematic map was to provide a broad lowing genome-editing overview of the progress on genome-editing in plants as techniques was used to well as the examination of associated of-target efects. Te validity of the included studies (critical appraisal) induce an alteration in Modrzejewski et al. Environ Evid (2019) 8:27 Page 7 of 33

was not assessed. However, in order to facilitate the deci- two) as well as provided in a searchable database that is sion on the potential of a subsequent systematic review, freely accessible on the web page https​://www.dialo​g-gea. data being indicative for the validity of an included study de/de/servi​ce/repos​itori​um. were extracted. Deviations from the systematic map protocol Data coding strategy Te methods used in this map deviate from the protocol Articles in which several genome-editing techniques in the following aspects: were applied, diferent plants were used or diferent genes were addressed have been subdivided in distinct stud- i. Contrary to what was stated in the protocol, the ies. While articles were screened for relevance at title/ recently developed base editing method was not abstract and full text level, the relevant data were fnally excluded. To identify all base editing studies the extracted at study level. second part of the search string (intervention) was Te data of each included study was extracted in one extended by the terms “base-editing” and “base row in the excel fle for the following superordinate editing”. categories: ii. Articles in other languages than English or Ger- man were included if further parts than title and 1. Bibliographic information (reference type, authors, abstract of the article like fgures or tables were in year of publication, title, abstract, keywords, peri- English or German and the provided information odical, issue number, page range, volume, DOI/ISBN, allowed for a defnite judgment of their relevance. corresponding author and the name of the country iii. In the course of data extraction we noticed that it the corresponding author is located). was not possible to properly categorize the pro- 2. Information answering secondary question one gress in research to the three classes indicated about traits modifed by genome-editing (genome- in the systematic map protocol [37]. Terefore, editing technique, plant species, sequence identi- we decided to classify the studies as either basic fer, trait, type of alteration, progress in research, key research or market-oriented application. To be topic). fagged as market-oriented, a study had to meet 3. Information answering secondary question two three criteria: about the occurrence of of-target efects due to the use of genome-editing (search for of-target efects, 1. Genome-editing was applied in an agricultural prediction of potential of-target efects (in silico), the crop. prediction method used, identifcation of potential 2. A trait was addressed that may be of interest for of-target sites, detection of of-target efects (biased/ commercialization (market-oriented trait). unbiased), detection method, amount of identifed 3. Te targeted trait is expressed in the edited plant of-target efects (biased/unbiased). grown. All studies that did not meet all three criteria Data were extracted according to the systematic map were classifed as basic research. protocol [37]. Data from a subset of 20 studies were vi. Contrary to what was stated in the protocol, no extracted by two reviewers independently to assess the EndNote database of all studies included in the sys- consistency of the extraction process across the review- tematic map was attached to this systematic map. ers. Discrepancies were discussed and clarifed within the Instead, one excel fle for each secondary ques- whole review team. Ten, the data of the other studies tion was provided as additional fle containing all were extracted by one reviewer and cross-checked by a included studies and the extracted data. second one to minimize the introduction of human error. In case of missing data, “no information” has been noted under the respective category. If data were in another lan- Results guage than English or German, it is stated as “language”. Review descriptive statistics Figure 1 presents the systematic mapping process of arti- Data mapping method cles and studies in a fow diagram. From January 1996 Te overview of research activities is provided in a nar- until May 2018, in total 15,703 records were identifed rative report and visualized in tables and fgures. In from ten bibliographic databases, Google Scholar, the addition, relevant studies and the extracted data are cata- targeted search on 47 company web pages, the USDA- logued in Additional fle 5 (to answer secondary question database “Am I regulated?” and the screening of 107 one) and Additional fle 6 (to answer secondary question review articles. After removing duplicates (n = 9521), Modrzejewski et al. Environ Evid (2019) 8:27 Page 8 of 33

Fig. 1 Flow diagram of the systematic mapping process explaining the selection of relevant articles and studies. This diagram follows ROSES guidance [40]

6182 articles remained and were screened on title/ literature on company websites and websites from gov- abstract level. Main reasons for exclusion at this stage ernmental authorities identifed another 31 further rel- were the application of genome-editing in animals and evant documents and web pages that were considered the absence of primary data. 941 articles passed the during data extraction. Out of these 555 records, a total inclusion criteria on title/abstract level or were rated amount of 1328 studies were extracted and formed the as “unclear” and remained included for full text screen- basis for this systematic map. A list of all studies and the ing. Te application of the inclusion criteria at full text extracted data to answer secondary question one is pro- level resulted in 524 relevant articles. Searching for grey vided in Additional fle 5. Additional fle 6 provides a list Modrzejewski et al. Environ Evid (2019) 8:27 Page 9 of 33

of all studies and the extracted data to answer second- case of grey literature, the study was accounted based on ary question two. A ROSES reporting form is included in the country the company is located at. Multiple assign- Additional fle 7. ments are possible if the corresponding author is afli- ated with institutions in diferent countries or if more General overview of the application of genome‑editing than one author was indicated being corresponding in model plants and crops author. Terefore, the sum of studies accounted for difer- Studies per year ent countries is higher (n = 1494) than the total amount In the mapping period between January 1996 and May of studies identifed (n = 1328). Asia is the leading con- 2018, a total amount of 1328 studies were identifed. As tinent when applying and publicizing genome-editing in shown in Fig. 2, the number of studies using TALENs, plants (n = 784 studies, 53%) followed by North America ZFN, ODM, MN and BE has remained on a relatively low (n = 508 studies, 34%), Europe (n = 189 studies, 13%), level. In contrast, the number of studies on CRISPR/Cas Australia (n = 6 studies, < 1%), South America (n = 4 has risen sharply soon after the system was applied for studies, < 1%) and Africa (n = 3 studies, < 1%) (Fig. 3). In the frst time in plants in 2013. Nearly 85% of the studies total, publications from 33 countries were identifed. As were published since 2015 indicating the rapid dissemi- shown in Fig. 3, China has a substantial lead in the num- nation and development of these techniques within the ber of studies (n = 599 studies, 40%) followed by the USA last few years. (n = 487 studies, 33%), Japan (94 studies, 6%) and Ger- As shown in Table 1, 26 studies used MN, 27 studies many (n = 88 studies, 6%). used ODM, 42 studies used BE, 73 studies used ZFN, 128 studies used TALENs and 1032 studies used CRISPR/ Genome‑editing applications in model plants and crops Cas. When using CRISPR/Cas, the most frequently used Around two-third of the studies (n = 907; 68%) were con- nuclease was Cas9 (n = 986) followed with a large dis- ducted on agricultural crops and one-third (n = 421; 32%) tance by Cas12a (also known as Cpf1) (n = 46). on model organisms. However, it is worth noting that rice is an important crop plant but it is also used as model Geographical distribution of genome‑editing studies species because of its genome size and because embryo- Te number of studies per country was calculated based genic rice cultures can be easily prepared, transformed on the country the corresponding author is located at. In and rapidly regenerated into fertile plants. In total, 51

500

450

400 CRISPR

350 TALENs

300 ZFN

ODM 250 MN 200 BE 150

100

50

0 1996 - 2012 2013 2014 2015 2016 2017 2018* Fig. 2 The number of genome-editing applications published per year. *Only January–May 2018; CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein, TALENs Transcription Activator-Like Efector Nucleases, ZFN Zinc-Finger Nucleases, ODM Oligo-Directed Mutagenesis, MN Meganucleases, BE base editing Modrzejewski et al. Environ Evid (2019) 8:27 Page 10 of 33

Table 1 Heat map showing number of studies performed with the diferent genome-editing techniques (rows) and the year the studies were published (columns) (1996–May 2018)

*Only January–May 2018 CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein, TALENs Transcription Activator-Like Efector Nucleases, ZFN Zinc- Finger Nucleases, ODM Oligo-Directed Mutagenesis, MN Meganucleases, BE base editing species and subspecies of diferent agricultural crops and except for a few nucleotides, was identical to the targeted 17 species/subspecies of model plants were under inves- sequence in which the DSB was introduced leading to a tigation, with the majority of studies focusing on rice DSB repair via homology-directed repair (SDN2). In 68 (n = 465) followed by the model organisms Arabidopsis studies (5%), a repair template was added that harbors a (n = 218) and tobacco (n = 107). Besides to these, tomato recombinant DNA sequence additional to the homolo- (n = 84) is most commonly studied followed by maize gous sequences and the DSB was repaired via homology- (n = 77), wheat (n = 63) and soybean (n = 53) (Fig. 4). directed repair (SDN3). Type of alteration in the plant genome Secondary question 1: “What are the traits modifed by genome‑editing in model plants as well as in crops As shown in Fig. 5, the majority of studies (n = 1223; 92%) describe the induction of point mutations or indels produced for agricultural production?” comparable to spontaneous mutations or undirected In total, 193 studies were allocated as market-oriented mutagenesis. Tis was mainly achieved with SDN1 applications. However, diferent scientists studied the (n = 1154) where no repair template was added and the same crop species and trait. Considering this, a total DSB was repaired by NHEJ. Additionally, the induction of amount of 99 diferent applications in 28 diferent plant point mutations (PM) using the ODM technique (n = 27) species remained. Tese market-oriented applications or BE (n = 42) leads to point mutations comparable to build the basis to answer secondary question 1. Figure 6 SDN1. Only 36 studies (3%) added a repair template that, categorizes the market-oriented applications to diferent Modrzejewski et al. Environ Evid (2019) 8:27 Page 11 of 33

Columbia 3 Argennia 1 Australia 6 USA 487 Canada 21 Germany 88 France 25 United Kingdom 21 Netherlands 15 Belgium 11 Ireland 5 Italy 4 Spain 4 Sweden 4 Denmark 3 Norway 3 Hungary 2 Austria 1 Greece 1 Poland 1 Turkey 1 China 599 Japan 94 South Korea 25 Israel 24 Saudi Arabia 18 Taiwan 9 India 5 Phillipines 5 Republic of Singapore 4 Pakistan 1 Kenya 2 Uganda 1

0 100 200 300 400500 600700 Fig. 3 Number of studies per country* in the systematic map database (grouped by continent; January 1996–May 2018). *Identifed by the corresponding author(s); South America: Grey; Australia: Black; North America: Orange; Europe: Blue; Asia: Green; Africa: Red Modrzejewski et al. Environ Evid (2019) 8:27 Page 12 of 33

Rice* 465 Arabidopsis 218 Tobacco* 107 Tomato* 84 Maize 77 Wheat* 63 Soybean 53 Algae* 49 Moss* 23 Potato 21 Canola 19 Barley 18 Clover* 16 Coon* 13 Poplar* 12 Sorghum* 10 Camelina 9 Brachypodium 8 Grapefruit 5 Cucumber 5 Apple 5 Grapevine 4 Parasponia 4 Orange 4 Alfalfa 3 Peanut 3 Cassava 3 Mushroom* 3 Cabbage* 3 Flax 2 Sage 2 Wild strawberry 2 Leuce 2 Watermelon 1 Pennycress 1 Sugarcane 1 Opium poppy 1 Mustard 1 Kiwifruit 1 Cacao 1 Fig 1 Dandelion 1 Citrange 1 Carrot 1 Banana 1 Bamboo 1 0100 200300 400500 Fig. 4 Total amount of genome-editing applications in crops and model plants (1996–May 2018). *Several species or subspecies were used when applying genome-editing Modrzejewski et al. Environ Evid (2019) 8:27 Page 13 of 33

Secondary question 2: “What is the available evidence 5% for the potential occurrence of associated of‑target efects 3% due to the use of genome‑editing in model plants as well as in crops produced for agricultural production?” SDN1/ PM/ BE In total, 252 studies from 161 articles were identifed in which the occurrence of of-target efects was assessed. SDN2 Table 8 maps the number of analyzed of-target efects for diferent genome-editing techniques and diferent plant SDN3 species. Most of the of-target analyses were conducted in CRISPR/Cas studies (n = 228) followed by TAL- 92% ENs studies (n = 9), BE studies (n = 9) and ZFN studies (n = 4). Solely in one ODM and in one MN study of-tar- get efects were investigated. Most of-target efects were analyzed in rice (n 93), followed by tomato (n 28), Fig. 5 Distribution of the type of alteration introduced by the = = application of genome-editing (January 1996–May 2018). SDN Site Arabidopsis (n = 23) and soybean (n = 15) (Table 8). directed nucleases, PM point mutation, BE base editing Of‑target efects considered for CRISPR/Cas‑systems More than 90% of the studies, in which of-target efects were assessed, were conducted with CRISPR/Cas. Fig- ure 8 provides an overview of the applied approaches to 5 6 identify of-target efects. In total, 228 CRISPR/Cas stud-

8 ies dealt with the analysis of of-target efects. 205 stud- 36 ies predicted potential of-target sites and 195 of these identifed potential of-target sites. Solely in 188 studies, these potential of-target sites were further analyzed for 16 the occurrence of of-target efects using biased detec- tion methods. In addition, 23 studies with already known potential of-target sites were assessed using biased 28 detection methods. So, in total, 211 studies analyzed potential of-target sites using biased detection methods. Agronomic value Food and feed quality Bioc stress tolerance Solely, nine studies searched for of-target mutations in Herbicide tolerance Industrial Abioc stress tolerance a completely unrestricted way using unbiased detection Fig. 6 Distribution of market-oriented applications of crops with methods. An overview of all identifed CRISPR/Cas stud- nutritionally, agriculturally or industrially relevant traits (January ies, in which of-target efects were addressed, is pro- 1996–May 2018) vided in Additional fle 6 including all extracted data.

Prediction of potential of‑target sites by CRISPR/Cas Diferent prediction tools can be used to identify poten- groups of traits. Most of the market-oriented applica- tial of-target sites. All of them have in common that tions (n 36) are related to an improved agronomic value = based on sequence alignment programs DNA-sequences (Table 2), followed by 28 applications with an improved are identifed in which unintended mutations could occur food and feed quality (Table 3). For biotic stress toler- due to high similarity between the targeted sequence and ance, 16 diferent applications were identifed (Table 4), the potential of-target site. However, the prediction of for herbicide tolerance eight applications (Table 5), for a potential of-target site is not equated with a real of- industrial utilization six applications (Table 6) and for target mutation. It has to be shown in a follow up step abiotic stress tolerance fve applications (Table 7). For by verifying the potential of-target site using biased more detailed information about the analyzed traits, see detection methods. 205 CRISPR/Cas studies searched for the respective tables. potential of-target sites. As shown in Fig. 9, many dif- As shown in Fig. 7 most of the market-oriented appli- ferent prediction tools were used to identify these sites. cations were applied in rice (n 29), followed by tomato = Mainly, three tools were used to predict potential of-tar- (n 16), maize (n 10), potato (n 6), wheat (n 6), = = = = get sites. BLAST was used 54 times, CRISPR-P 51 times soybean (n 4) and canola (n 4). In 21 other agricul- = = and CasOFF-Finder 31 times. 12 other prediction tools tural relevant crops, one or two market-oriented applica- were used in 31 studies (for detailed information see tions were identifed. Modrzejewski et al. Environ Evid (2019) 8:27 Page 14 of 33

Rice 29 Tomato 16 Maize 10 Potato 6 Wheat 6 Soybean 4 Canola 4 Switchgrass 2 Cucumber 2 Coon 2 Wild strawberry 1 Tobacco 1 Sugarcane 1 Sage 1 Poplar 1 Pennycress 1 Peanut 1 Orange 1 Opium poppy 1 Mushroom 1 Leuce 1 Grapevine 1 Grapefruit 1 Flax 1 Cassava 1 Camelina 1 Cacao 1 Alfalfa 1 0510 15 20 25 30 35 Fig. 7 Total amount of genome-editing applications in crops with market-oriented traits (January 1996–May 2018)

Additional fle 6). 41 studies did not provide any details no information about the used detection method was about the used tool(s). Tree studies used two diferent provided. prediction tools to identify potential of-target sites. Taking all CRISPR/Cas studies together, 1738 difer- Te number of predicted of-target sites varies widely ent potential of-target sites were analyzed using targeted between studies from zero to 4265. Several reasons for sequencing. Of-target efects were identifed in 55 of this broad heterogeneity exist, which were not extracted these sites, indicating that in around 3% of the analyzed in detail within this map but will be elucidated in the sequences of-target mutations were detected. In another discussion. six studies, no information was provided about the amount of analyzed of-target sites but of-target muta- Detection of of‑target efects—biased tions were not identifed either. Targeted sequencing to pre-selected sites was applied in Considering the diferent plant species, most of the 211 CRISPR/Cas studies to detect of-target efects. As CRISPR/Cas studies using biased detection methods shown in Fig. 8, the predicted of-target sites were fre- were conducted in rice (n = 77), followed by tomato quently analyzed for the occurrence of of-target efects (n = 23), Arabidopsis (n = 21), diferent moss spe- using biased detection methods. In a few studies, poten- cies (n = 13) and soybean (n = 12) (Table 9). In rice, a tial of-target sites were already known and analyzed total amount of 291 potential of-target sites were ana- without using sequence alignment programs a priori. Fig- lyzed and in 25 of these sites, of-target mutations were ure 10 displays the diferent detection methods applied detected. In contrast, studies conducted in tomatoes to identify of-target mutations. In the large majority of solely reported the identifcation of one of-target muta- studies, of-target efects were detected using a PCR fol- tion when analyzing 222 potential of-target sites. lowed by sequencing (n = 137). Only a few studies used In one study, a diferent approach was chosen to assess the detection methods RE-PCR assay (n = 16), targeted the occurrence of of-target efects [175]. In this study, a deep sequencing (n = 15), enzyme mismatch cleavage series of mismatches were introduced at the sgRNA fol- assay (n = 13) and CAPS analyses (n = 13). In 11 studies, lowed by analyzing whether the targeted sequence was Modrzejewski et al. Environ Evid (2019) 8:27 Page 15 of 33

Table 2 Genome-editing in plants for modifying agronomically relevant traits (1996–May 2018) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Canola Christian-Albrechts-University of Kiel, Increased yield Increased shatter resistance to avoid CRISPR/Cas9 [41] Germany seed loss during mechanical SDN1 harvest Canola Huazhong Agricultural University, Increased yield Increased seeds number per husk, CRISPR/Cas9 [42] China higher seed weight SDN1 Cotton Anhui Agricultural University, China; Growth characteristics Improved root growth under high CRISPR/Cas9 [43] Chinese Academy of Agricultural and low nitrogen conditions SDN1 Sciences, China Cucumber Chinese Academy of Agricultural Sci- Growth characteristics Only female fowers CRISPR/Cas9 [44] ences, China SDN1 Lettuce University of California, USA Increased yield Germination at high temperature CRISPR/Cas9 [45] SDN1 Maize Benson Hill Biosystems, USA Increased yield Increased photosynthesis efciency Meganuclease [46] SDN3 Maize University of Wisconsin, USA Growth characteristics Early fowering under long day condi- CRISPR/Cas9 [47] tions SDN1 Maize DuPont Pioneer, USA Growth characteristics Male sterility CRISPR/Cas9 [48, 49] SDN1 University of Science and Technology CRISPR/Cas9 [4] Beijing, China; Beijing Solidwill Sci- SDN1 Tech Co. Ltd, China Chinese Academy of Sciences, China CRISPR/Cas9 [50] SDN1 Maize Syngenta Seeds, USA Growth characteristics Haploid induction TALENs [51] SDN1 Potato Cellectis Plant Science, USA Storage characteristics Improved cold storage and process- TALENs [52] ing traits (reduced sugars/reduced SDN1 levels of acrylamide) Rice Chinese Academy of Sciences, China Increased yield Altered grain number per panicle CRISPR/Cas9 [53] SDN1 National Rice Research Institute, China CRISPR/Cas9 [54] SDN1 Rice Chinese Academy of Sciences, China Increased yield Seed size/increased seed weight CRISPR/Cas9 [53] SDN1 Anhui Academy of Agricultural Sci- CRISPR/Cas9 [55] ences, China SDN1 Fudan University, China CRISPR/Cas9 [56] SDN1 Yangzhou University, China CRISPR/Cas9 [57] SDN1 Agronomy College of Henan Agricul- CRISPR/Cas9 [58] tural University, China SDN1 Chinese Academy of Agricultural Sci- CRISPR/Cas9 [59] ences, China; Yangzhou University, SDN1 China Rice Chinese Academy of Sciences, China Growth characteristics Increased plant height, improved CRISPR/Cas9; [53, 60] tiller-production, erect panicle, SDN1 Wuhan Institute of Bioengineering; increased biomass CRISPR/Cas9 [60] Huazhong Agricultural University, SDN1 China Sichuan Agricultural University CRISPR/Cas9 [61] SDN1 Chinese Academy of Agricultural Sci- CRISPR/Cas9 [59] ences, China; Yangzhou University, SDN1 China Modrzejewski et al. Environ Evid (2019) 8:27 Page 16 of 33

Table 2 (continued) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Rice Chinese Academy of Agricultural Growth characteristics Early maturing CRISPR/Cas9 [62] Sciences, China; Jangsu Academy of SDN1 Agricultural Sciences, China Rice Kyung Hee University, South Korea Growth characteristics Male sterility CRISPR/Cas9 [63] SDN1 Shanghai Jiao Tong University, China CRISPR/Cas9 [64] SDN1 South China Agricultural University, CRISPR/Cas9 [65, 66] China SDN1 Sichuan Agricultural University, China CRISPR/Cas9 [67, 68] SDN1 Rice Chinese Academy of Sciences, China; Increased yield Regulation of pollen tube growth CRISPR/Cas9 [69] University of Chinese Academy of SDN1 Sciences, China Rice China Agricultural University, China Storage characteristics Increased seed storage TALENs [70] SDN1 Rice China National Rice Research Institute, Increased yield Increased seed setting rate CRISPR/Cas9 [71] China; China Three Gorges Univer- SDN1 sity, China Rice Anhui Academy of Agricultural Sci- Increased yield Longer panicle CRISPR/Cas9 [55] ences, China SDN1 Rice Nanjing Agricultural University, China Increased yield Grain yield, regulation of seed devel- CRISPR/Cas9 [72] opment SDN1 Rice Chinese Academy of Sciences, China Growth characteristics Decreased plant height CRISPR/Cas9 [73] SDN1 Syngenta Biotechnology, China CRISPR/Cas9 [74] SDN1 Rice Wuhan Institute of Bioengineer- Increased yield Increased nitrogen utilization ef- CRISPR/Cas9 [60] ing, China; Huazhong Agricultural ciency SDN1 University, China Rice Hunan Normal University, China Growth characteristics Regulation of seed dormancy, stoma- CRISPR/Cas9 [75] tal opening, plant developmental, SDN1 abiotic stress tolerance and leaf senescence Soybean Chinese Academy of Agricultural Sci- Growth characteristics Late fowering CRISPR/Cas9 [76] ences, China SDN1 Switchgrass Iowa State University, USA Growth characteristics Bushy phenotype CRISPR/Cas9 [77] SDN1 Tomato National Food Research Institute, Increased yield Regulating fruit ripening CRISPR/Cas9 [78] Japan SDN1 Tomato University of Minnesota, USA Growth characteristics Bigger seedlings TALENs [79] SDN1 Tomato Cold Spring Harbor Laboratory, USA; Growth characteristics Early fowering CRISPR/Cas9 [80] Max Planck Institute for Plant Breed- SDN1 ing Research, Germany; Université Paris-Scalay, France Tomato University of Florida, USA Growth characteristics Easy separation of fruit and stem CRISPR/Cas9 [81] SDN1 Tomato Cold Spring Harbor Laboratory, USA Increased yield Fruit size CRISPR/Cas9 [82] SDN1 Tomato Cold Spring Harbor Laboratory, USA Increased yield Highly branched inforescence and CRISPR/Cas9 [82] formation of multiple fowers SDN1 Tomato Weizmann Institute of Science, Israel Growth characteristics Yellow fruit color CRISPR/Cas9 [83] SDN1 Weizmann Institute of Science, Israel CRISPR/Cas9 [84] SDN 3 Modrzejewski et al. Environ Evid (2019) 8:27 Page 17 of 33

Table 2 (continued) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Tomato Weizmann Institute of Science, Israel Growth characteristics Orange fruit color CRISPR/Cas9 [84] SDN3 Tomato Academy of Agriculture and Forestry Growth characteristics Pink fruit color CRISPR/Cas9 [85] Sciences; Chinese Academy of Sci- SDN1 ences, China Wheat Kansas State University, USA Increased yield Bigger grains, increased grain weight CRISPR/Cas9 [86] SDN1 Chinese Academy of Sciences, China CRISPR/Cas9 [87] SDN1 Wild strawberry University of Maryland, USA Growth characteristics Faster seedling growth CRISPR/Cas9 [88] SDN1 TALENs Transcription Activator-Like Efector Nucleases, CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, SDN Site directed nucleases successfully mutated despite the mismatch(es) between specifc advantages and disadvantages and could the sgRNA and the targeted sequence. Additionally, in afect the occurrence of of-target efects [26, 32]. this study, the of-target patterns between two PAMs iv. Te number of hypothetically tolerated mismatches (NGG and NAG) were compared. 22 times the sgRNA between the target sequence and the potential was designed in a way that it contained one mismatch to of-target sites which is an exercise in combina- the targeted sequence. 15 of these altered sgRNA induced torics. Ali et al. [176] identifed a total amount of a DSB at the targeted sequence. 14 times the sgRNA con- 4265 potential of-target sites in the model organ- tained two mismatches to the targeted sequence and four ism Nicotiana benthamiana for a CRISPR/Cas9 of these sgRNA induced a DSB in the targeted sequence. sgRNA. To identify candidate of-target sites, the Moreover, eight times the sgRNA contained three mis- genome was screened allowing one to seven mis- matches. In these cases, no mutation was identifed in matches to the target sequence. Hence, the more the targeted sequence [175]. According to the predic- mismatches are tolerated in prediction the higher tion of of-target efects, the summary provided here is the number of potential of-target sites (in the does not allow any conclusions to be drawn due to broad paper: one, two or three mismatches tolerated: No heterogeneity. potential of-target sites, four mismatches toler- ated: One potential of-target site, fve mismatches Heterogeneity in CRISPR/Cas‑studies tolerated: 60 potential of-target sites, six mis- regarding the evidence how to predict and detect matches tolerated: 515 potential of-target sites, potential of‑target efects seven mismatches tolerated: 3689 potential of-tar- Te number of potential of-target sites called “identifed” get sites). Tis indicates that the number of poten- by the respective authors varies widely between zero and tial of-target sites strongly depends on the number 4265. Several reasons were identifed that could explain of hypothetically tolerated mismatches. Trough- this broad heterogeneity: out the available literature, the number of tolerated mismatches predetermined by the researchers was i. In total, potential of-target sites were investigated very heterogeneous ranging up to 13 mismatches in over 30 diferent plant species and subspecies. [177]. Te diferent genome sizes and the diferent num- v. Individual studies also deviate between diferent ber of chromosome sets of the individual plants structural assumptions when determining poten- vary widely which infuence the number of poten- tial of-target sites. In most studies, a potential of- tial of-target sites. target was only counted as such if a PAM followed ii. To identify potential of-target sites in CRISPR/ the potential of-target site. However, in some stud- Cas-studies 15 diferent prediction tools were ies, potential of-target sites were assigned as such applied. without being followed by a PAM (e.g. [44, 178]), iii. For the detection of of-target efects, various although at these sites no DSB can be induced with methods have been used, but all of them show their the specifc CRISPR-nucleases used. Modrzejewski et al. Environ Evid (2019) 8:27 Page 18 of 33

Table 3 Genome-editing in plants for improved food and feed quality (1996–May 2018) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Alfalfa Calyxt, Inc., USA Product quality Reduced lignin content TALENs [89] SDN1 Camelina sativa Montana State University, USA Product quality Increased levels of oleic acid and CRISPR/Cas9 [90] α-linolenic acid SDN1 University Nebraska, USA Increased levels of oleic acid, decreased CRISPR/Cas9 [91] levels of fatty acids SDN1 Université Paris-Saclay, France Increased levels of oleic acid, decreased CRISPR/Cas9 [92] levels of fatty acids SDN1 Kansas State University, USA Lower oil content CRISPR/Cas9 [93] SDN1 Canola Tamagawa University, Japan Product quality Altered fatty acid composition CRISPR/Cas9 [94] SDN1 Maize Du Pont Pioneer, USA; Product quality Waxy corn, improved starch production CRISPR/Cas9 [95] SDN1 Chinese Academy of Agricultural Sci- Waxy phenotype, abolition of amylose CRISPR/Cas9 [96] ences, China SDN1 Maize Agrivida, USA Product quality Higher levels of starch in their leaves Meganucleases [97] and stalks SDN1 Maize Dow AgroScience, USA Product quality Reduced phytate production herbi- ZFN [98, 99] cide tolerance + SDN3 Mushroom Penn State University, USA Product quality Non-browning mushroom CRISPR/Cas9 [100] SDN1 Opium poppy Cankiri Karatekin University, Turkey; Product quality Reduced morphine and thebaine CRISPR/Cas9 [101] Dokuz Eylul University, Turkey content SDN1 Peanut Guangdong Academy of Agricultural Product quality Increased oleic acid content, decreased TALENs [102] Sciences, China linoleic acid content SDN1 Potato Calyxt, USA Product quality Non-browning potato TALENs [103] SDN1 Potato Simplot Plant Science, USA Product quality Reduced black spottiness TALENs [104] SDN1 Potato RIKEN Center for Sustainable Resource Product quality Reduction of harmful ingredients (gly- TALENs [105] Science, Japan; Chiba University, Japan coalkaloids) SDN1 Kobe University, Japan Complete abolition of glycoalkaloids CRISPR/Cas9 [106] (bitter taste) SDN1 Rice Chinese Academy of Sciences, China Product quality Fragrant rice TALENs [107] SDN1 Chinese Academy of Agricultural Sci- CRISPR/Cas9 [59] ences, China; Yangzhou University, SDN1 China Rice Chinese Academy of Agricultural Sci- Product quality Increased contents beneftting human CRISPR/Cas9 [108] ences, China; University of California, health (increased amylose content) SDN1 USA Rice Huazhong Agricultural University, China Product quality Reduced contents harming human CRISPR/Cas9 [109] health (arsenic content) SDN1 Sun Yat-sen University, China CRISPR/Cas9 [110] SDN1 Rice National Agriculture and Food Research Product quality Altered fatty acid composition CRISPR/Cas9 [111] Organization, Japan SDN1 Rice Université Montpellier, France Product quality Reduced contents harming human CRISPR/Cas9 [112] health (cesium content) SDN1 Rice Hunan Agricultural University, Hunan Product quality Reduced contents harming human CRISPR/Cas9 [113] Hybrid Rice Research Center, Normal health (cadmium content in plants) SDN1 University, China Rice Chinese Academy of Sciences, Shanghai, Product quality Waxy rice CRISPR/Cas9 [114] China; Purdue University, West Lafay- SDN1 ette, USA Modrzejewski et al. Environ Evid (2019) 8:27 Page 19 of 33

Table 3 (continued) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Sage Second Military Medical University, Product quality Reduced phenolic acid content CRISPR/Cas9 [115] China SDN1 Soybean Cellectis plant science Inc., USA/Calyxt, Product quality High oleic content, low linoleic content TALENs [21, 116–118] USA SDN1 Tomato Agricultural Research Organization, Product quality Seedless tomato CRISPR/Cas9 [119] Israel SDN1 Tokushima University, Japan CRISPR/Cas9 [120] SDN1 Tomato University of Tsukuba, Japan Product quality Increased contents beneftting human CRISPR/Cas9 [121] health (increased GABA content) SDN1 China Agricultural University, China CRISPR/Cas9 [122] SDN1 Tomato China Agricultural University, China Product quality Increased contents beneftting human CRISPR/Cas9 [123] health (increased lycopene content) SDN1 Tomato Xinjiang Academy of Agricultural Sci- Product quality Improved shelf life CRISPR/Cas9 [124] ence, China SDN1 Wheat Calyxt, Inc., USA Product quality Increased nutritional value TALENs [125] SDN1 Wheat Instituto de Agricultura Sostenible (IAS- Product quality Reduced gluten content CRISPR/Cas9 [126] CSIC), Spain; University of Minnesota, SDN1 USA Wheat (durum) Instituto de Agricultura Sostenible Product quality Reduced gluten content CRISPR/Cas9 [126] (IAS-CSIC), Spanien; University of Min- SDN1 nesota, USA TALENs Transcription Activator-Like Efector Nucleases, CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, ZFN Zinc- Finger Nucleases, SDN Site directed nucleases

vi. In some studies, the sgRNA was selected tak- Of‑target efects considered for TALENs systems ing into account that no potential of-target efect In the period until May 2018, nine TALENs studies should occur. Care was taken that, apart from the were identifed addressing of-target efects. Additional target sequence, no other sites in the genome pos- fle 6 provides an overview of these studies including sess a similar sequence that could result in an of- all extracted data according to the systematic map pro- target mutation. Tus, the predicted number of tocol. Figure 11 maps the diferent approaches used to potential of-target sites is lower or zero in these analyze of-target efects. Five studies predicted poten- studies compared to studies, in which potential tial of-target sites. Two times, the TAL Efector Nucleo- of-target sites were not considered a priori in the tide Targeter 2.0 was used and one time each of the tools sequence selection. PROGNONS, kmasker and Arabidopsis Information Resource PatMatch. Te number of identifed potential of-target sites varies widely between zero and 18. In one Detection of of‑target efects—unbiased study, no precise information was given and it was just Nine CRISPR/Cas studies were identifed using whole indicated that many potential of-target sites were identi- genome sequencing (WGS) as an unbiased detection fed. Te four studies that identifed potential of-target method to identify genome-wide of-target efects (see sites investigated these for the occurrence of of-target Additional fle 6). No of-target mutations were detected efects using biased detection methods. In addition, three in any of these studies. One study compared both biased studies with already known potential of-target sites and unbiased detection methods. While using unbiased assessed these sites. Diferent tools were used to exam- detection methods, no of-target efects were detected, ine whether of-target efects occurred. Two studies used but biased methods detected one of-target [179]. An enzyme mismatch cleavage assay and one study each explanation for this could be that WGS is able to detect PCR + Sequencing, PCR + CAPS and RE-PCR assay. In higher frequency of-target efects only, but lacks the two studies, no detailed information about the applied sensibility required to detect of-target mutations in bulk detection method was provided. In total, 31 potential of- population [26]. target sites were analyzed for the occurrence of of-target Modrzejewski et al. Environ Evid (2019) 8:27 Page 20 of 33

Table 4 Genome-editing in plants for increased resistance to biotic stress (1996–May 2018)

Plant Developer, producer, country Trait Specifcation Technological specifcation References

Cacao Pennsylvania State University, USA Fungal resistance Resistance to Phytophthora tropicalis CRISPR/Cas9 [127] SDN1 Cucumber Volcani Center, Israel Virus resistance Immunity to cucumber vein yellow- CRISPR/Cas9 [22] ing virus (Ipomovirus) infection SDN1 and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W Grapefruit University of Florida, USA Bacterial resistance Resistance to citrus canker CRISPR/Cas9 [128, 129] SDN1 Grapevine Northwest A&F University and Minis- Fungal resistance Resistance to Botrytis cinerea CRISPR/Cas9 [130] try of Agriculture, China SDN1 Maize Du Pont Pioneer, USA Fungal resistance Resistance to Northern Leaf Blight CRISPR/Cas9 (Cisgenesis) [131] (NLB) SDN3 Orange Chinese Academy of Agricultural Bacterial resistance Resistance to citrus canker CRISPR/Cas9 [132] Sciences and National Center for SDN1 Citrus Variety Improvement; South- west University, China Rice Chinese Academy of Agriculture, Fungal resistance Resistance to rice blast CRISPR/Cas9 [133] China SDN1 Rice Iowa State University, USA Bacterial resistance Resistance to bacterial blight CRISPR/Cas9 [134] SDN1 IRD-CIRAD-Université, France TALENs [135] SDN1 Iowa State University, USA TALENs [136] SDN1 National University of Singapore, TALENs [137] Singapore SDN1 Chinese Academy of Sciences, China TALENs [138] SDN1 National Center for Plant Gene CRISPR/Cas9 [139] Research, China; Sichuan Agricul- SDN1 tural University, China Rice Iowa State University, USA Fungal resistance Resistance to powdery mildew TALENs [140] SDN1 Rice Shanghai Jiao Tong University, China; Bacterial resistance Resistance to the pathogen Xoc TALENs [141] Yunnan Academy of Agricultural RS105 SDN1 Sciences, China Rice Sichuan Agricultural University, China Bacterial resistance/ Resistance to bacterial blight and CRISPR/Cas9 [61] fungal resistance rice blight SDN1 Rice International Rice Research Institute Virus resistance Resistance to rice tungro disease CRISPR/Cas9 [142] (IRRI), Philippines (RTD) SDN1 Tomato Max Planck Institute for Develop- Fungal resistance Resistance to powdery mildew CRISPR/Cas9 [143] mental Biology, Germany; Norwich SDN1 Research Park, UK Tomato King Abdullah University of Science Virus resistance Resistance to tomato yellow leaf virus CRISPR/Cas9 [144] and Technology, Saudi Arabia SDN1 Tomato University of California, USA Bacterial resistance Resistance to diferent pathogens CRISPR/Cas9 [145] including P. syringae, P. capsici and SDN1 Xanthomonas spp. Wheat Chinese Academy of Sciences, China Fungal resistance Resistance to powdery mildew TALENs [25] SDN1 Chinese Academy of Sciences, China CRISPR/Cas9 [146] SDN1 Calyxt, Inc., USA TALENs [147] SDN1

TALENs Transcription Activator-Like Efector Nucleases, CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, SDN Site directed nucleases Modrzejewski et al. Environ Evid (2019) 8:27 Page 21 of 33

Table 5 Genome-editing for generating herbicide tolerant plants (1996–May 2018) Plants Developer, producer, country Traita Specifcation Technological specifcation References

Canola Cibus, Canada; Cibus, USA Herbicide tolerance – ODM [23] Bayer BioScience N.V., Belgium [148] Cassava Donald Danforth Plant Science Center, St. Louis, USA Herbicide tolerance – CRISPR/Cas9 [149] SDN3 Cotton Bayer CropScience N.V., Belgium Herbicide tolerance – Mega-nucleases [150] SDN3 Flax Cibus, USA Herbicide tolerance – CRISPR/Cas9 SDN1 [151] Maize DuPont Pioneer, USA Herbicide tolerance – CRISPR/Cas9 [48, 49] SDN1, SDN2, SDN3 Dow AgroScience, USA ZFN SDN3 [99, 152] Pioneer Hi-Bred International, USA ODM [153, 154] Potato Michigan State University, USA Herbicide tolerance – CRISPR/Cas9, TALENs SDN2 [155] Rice Chinese Academy of Sciences, China Herbicide tolerance – CRISPR/Cas9 SDN2 [156] Chinese Academy of Sciences, China; Huazhong Agri- CRISPR/Cas9 SDN2 [157] cultural University, China; University of California San Diego, USA Zhejiang University, China TALENs SDN2 [158] Tohoku University, Japan ODM [159] Kobe University, Japan; University of Tsukuba, Japan BE [160, 161] King Abdullah University of Science and Technology, CRISPR/Cas9 SDN2 [162] Saudi Arabia Soybean DuPont Pioneer, USA Herbicide tolerance – CRISPR/Cas9 SDN2 [163] CRISPR/Cas9 SDN3 [164] a No detailed breakdown regarding chemical agents TALENs Transcription Activator-Like Efector Nucleases, CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, ZFN Zinc- Finger Nuclease, ODM Oligo-Directed Mutagenesis, SDN Site directed nucleases, BE base editing

Table 6 Genome-editing in plants for industrial utilization (1996–May 2018) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Pennycress Illinois State University, USA Product quality Altered oil composition CRISPR/Cas9 [165] SDN1 Poplar University of Georgia, USA Product quality Stem wood discoloration due CRISPR/Cas9 [166] to lignin reduction SDN1 Potato Swedish University of Agricultural Sci- Product quality Improved starch quality CRISPR/Cas9 [167] ences, Sweden SDN1 Sugarcane University of Florida, USA Product quality Reduced lignin content TALENs [168, 169] SDN1 Switchgrass Noble Research Institute, USA Product quality Reduced lignin content CRISPR/Cas9 [170] Tobacco North Carolina State University, USA Product quality Reduced nicotine content Meganucleases [171] SDN1

CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, TALENs Transcription Activator-Like Efector Nucleases, SDN Site directed nucleases efects and one of these sites contained an of-target of these mutations cannot be ruled out to be spontane- mutation. In one study, an unbiased search for of-tar- ous ones or sequencing errors [180]. get efects was conducted [180]. Using Whole Genome Sequencing (WGS), three of-target mutations were iden- Of‑target efects considered for Zinc‑Finger Nucleases tifed which were not present in the wild-type sample. systems However, the of-target sequences showed no similarity Four studies dealt with the analyses of of-target efects to the TALENs binding sites. Terefore, the occurrence when applying ZFN in plants. Detailed information is Modrzejewski et al. Environ Evid (2019) 8:27 Page 22 of 33

Table 7 Genome-editing in plants to improve tolerance to abiotic stress (1996–May 2018) Plant Developer, producer, country Trait Specifcation Technological References specifcation

Maize Ghent University, Belgium; Center for Plant Systems Biology, Drought tolerance – CRISPR/Cas9 [172] Belgium; Jomo Kenyatta University of Agriculture and Technol- SDN1 ogy, Kenia DuPont Pioneer, USA CRISPR/Cas9 [24, 164] SDN3 Rice Anhui Academy of Agricultural Sciences, China Salt tolerance – CRISPR/Cas9 [20] SDN1 Rice Huazhong Agricultural University, China Arsenic tolerance – CRISPR/Cas9 [109] SDN1 Sun Yat-sen University, China CRISPR/Cas9 [110] SDN1 Soybean USDA-ARS, USA Drought and salt tolerance – CRISPR/Cas9 [173] SDN1 Wheat Montana State University, USA Drought tolerance – CRISPR/Cas9 [174] SDN1

CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9, SDN Site directed nucleases provided in Additional fle 6. All studies identifed puta- no information). According to the other genome-editing tive of-target sites that are most closely related to the techniques, the number of potential of-target sites varies target sequences. Two times, the database PLantGDB widely between one and nine. In a second step, all pre- was used to identify potential of-target sites and two dicted potential of-target sites were sequenced for the times no detailed information was provided. In total, 10 occurrence of of-target efects using PCR + sequenc- potential of-target sites were detected which were then ing method (n = 6), targeted deep sequencing (n = 2) or screened for the occurrence of of-target efects. No of- enzyme mismatch cleavage assay (n = 1). One of-target target mutations were detected in any of these analyzed, mutation was identifed. potential of-target sites. Discussion Of‑target efects considered for Meganuclease systems Market oriented applications of genome‑editing Solely one MN study was identifed that analyzed of- Tis map documents the state of evidence for the applica- target efects (Additional fle 6). In this study, a homolo- tion of genome-editing as a new tool for the modifcation gous sequence to the target one was identifed that difers of plant traits and the associated potential occurrence solely in two nucleotides. However, when screening this of of-target efects. Te publication rate of primary potential of-target site no modifcation was identifed. research has risen sharply since the CRISPR/Cas tech- nique was frst applied in plants in 2013. It is worth men- Of‑target efects considered for Oligonucleotide‑Directed tioning that in total primary studies from 33 countries Mutagenesis systems were identifed but nearly three quarter of these studies One study addressed the occurrence of of-target originate from either China (40%) or the USA (33%). For efects in rice using the ODM technique (Additional comparison, Japan and Germany published around 6% of fle 6). Beside the targeted Acetolactate synthase (ALS) the studies each and no other country contributed more sequence, the whole coding region of ALS gene was than 2% of the total. Summarizing the number of pub- sequenced but no further mutation was detected. lished studies by continent, more than 50% of the studies were conducted in an Asian country (53%), around one- Of‑target efects considered for base editing systems third in North American countries (34%) and only 13% in In total, nine BE studies analyzed the occurrence of of- European countries. As the genome-editing techniques, target efects (Additional fle 6). However, one study especially CRISPR/Cas, were just recently developed, could only rarely be evaluated due to language barriers the large majority of the existing applications represent [181]. Te approach used to identify of-target efects basic research. Nevertheless, almost 100 diferent appli- was similar in all studies (Fig. 12). In a frst step, potential cations aimed to produce benefcial agricultural traits in of-target sites were predicted using diferent tools (2× 28 diferent agricultural crops. We determined that the CRISPR-GE tool, 2× CRISPR-P, 1× CasOf-Finder, 3× majority of such “market-oriented” applications have Modrzejewski et al. Environ Evid (2019) 8:27 Page 23 of 33

Table 8 Overview of of-target studies in relation to diferent genome-editing techniques and plant species (January 1996–May 2018)

CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein, TALENs Transcription Activator-Like Efector Nucleases, ZFN Zinc- Finger Nuclease, ODM Oligo-Directed Mutagenesis, MN Meganucleases, BE base editing

been carried out in economically important crops such chain and could contribute to food security and the envi- as maize, rice, wheat and soybeans, but less economi- ronmental management. To confrm this hypothesis a cally important crops such as cucumber, lettuce, peanut systematic review would be required to quantify such or grapefruit have been worked on as well. Te market- efects. oriented applications address several breeding objectives including yield improvement, improved growth charac- Of‑target efects of genome‑editing teristics, improved food and feed quality, tolerances to 252 studies investigated the occurrence of of-target biotic and abiotic stress, herbicide tolerance and indus- efects following a genome-editing application in plants. trial utilization. Tis indicates that genome-editing is Most of these studies were performed with the genome- able to address benefcial traits for the agricultural value editing technique CRISPR/Cas (n = 228), whereas only a Modrzejewski et al. Environ Evid (2019) 8:27 Page 24 of 33

Fig. 8 Overview of the used approaches to assess of-target efects in CRISPR/Cas studies. All numbers represent the amount of studies conducted for diferent approaches. In some studies, several approaches were used to analyze of-targets

occurrence of of-target efects. Te biased method sug- few studies used TALENs (n = 9), BE (n = 9), ZFN (n = 4), gests a very broad data basis, but as shown in the result MN (n = 1) or ODM (n = 1). Reasons for these fndings are that the total number of studies in which CRISPR/ section, individual studies are very heterogeneous in Cas was applied is much larger compared to the other their structure and design. Heterogeneity was identifed genome-editing techniques. Additionally, CRISPR/Cas regarding the plant species, the CRISPR-variant, the pre- is more susceptible to of-target efects compared to diction tools and detection methods used, the amount of other nuclease techniques such as ZFN and TALENs, as tolerated mismatches and the chosen sgRNA. In order to it works as a monomer, whereas ZFN and TALENs work allow any conclusions to be drawn about the occurrence as dimers [182]. In addition, the sgRNA used in CRISPR/ of of-target efects a more in-depth analysis e.g. by a sys- Cas-applications is able to tolerate several mismatches tematic review is mandatory. leading to the induction of a DSB at a site in the DNA that is similar but not identical to the targeted sequence Knowledge gaps [32]. In most CRISPR/Cas studies biased detection Regarding secondary question 2, several topics have been methods were used, meaning that potential of-target identifed representing knowledge gaps where no stud- sites were frst predicted using bioinformatics programs ies or only a small number of studies exist. A knowledge followed by targeted sequencing of these sites for the gap exists for the analysis of of-target efects in TALENs, ZFN, MN, ODM and BE. Only nine studies have been Modrzejewski et al. Environ Evid (2019) 8:27 Page 25 of 33

60 54 51 50 41 40 31 31 30

20

10

0 BLASTCRISPR-PCasOFF-Finder Others No informaon Fig. 9 Tools used to predict potential of-target sites in 205 CRISPR/Cas-studies (1996–May 2018). Three studies employed two tools

160 137 140

120

100

80

60

40 16 15 20 13 13 9 11

0 PCR + RE-PCR assayTargeted deep Enzyme PCR, CAPSOthersNo Sequencing sequencing mismatch informaon cleavage assay Fig. 10 Biased detection methods used to identify of-target efects in CRISPR/Cas studies. Three studies employed two tools. PCR polymerase chain reaction, RE–PCR restriction enzyme–polymerase chain reaction, CAPS cleaved amplifed polymorphic sequences identifed analyzing the occurrence of of-target efects in has great potential to support plant breeding, though it TALENs and BE studies. Te amount of of-target studies is not broadly established yet. It is based on the CRISPR/ for ZFN (n = 4), ODM (n = 1) and MN (n = 1) were even Cas9 system but enables exchanging individual base pairs lower. For a more in-depth of-target analysis regarding at specifc sites without inducing a DSB. Our map shows these techniques, further research and more primary that only a few BE studies addressed the occurrence of studies are needed. However, the CRISPR/Cas technique of-target efects so far. Te likely reason is that BE is a is applied much more frequently because of efciency, “young” method in plants, and its frst publication was time saving and cost-efectiveness compared to TALENs, in 2017. Further research about BE including the analy- ZFN, ODM and MN [182]. Terefore, it is questionable sis of of-target efects is highly recommended. Another what additional research efort is justifed. BE difers as it knowledge gap afecting all genome-editing techniques is Modrzejewski et al. Environ Evid (2019) 8:27 Page 26 of 33

Table 9 Overview of the amount of studies analyzing potential of-target sites, the amount of analyzed potential of- target sites and the amount of identifed of-target efects using biased detection methods in diferent plant species Plant species Amount of studies analyzing preselected Amount of analyzed potential of- Amount of identifed of- potential of-target sites target sites—biased target efects—biased

Rice 77 296 25 Tomato 23 213 1 Arabidopsis 21 229 4 Moss 13 58 0 Soybean 12 106 6 Tobacco 9 50 1 Wheat 8 85 6 Maize 7 23 2 Algae 4 357 0 Canola 4 73 0 Cotton 4 58 0 Orange 3 21 6 Grapevine 3 13 0 Poplar 3 6 0 Grapefruit 2 15 0 Cucumber 2 9 0 Clover 2 3 0 Lettuce 1 91 0 Cacao 1 9 0 Flax 1 8 0 Barley 1 4 1 Kiwifruit 1 4 0 Camelina 1 3 0 Citrange 1 3 0 Watermelon 1 3 0 Cabbage 1 2 1 Carrot 1 1 1 Potato 1 1 0 Sage 1 1 0 Wild strawberry 1 1 0 Alfalfa 1 No information 0

the genome-wide analysis of of-target efects in a com- • Genome composition, size and ploidy level of difer- pletely unrestricted way. Only nine studies addressed this ent plant species. aspect using the CRISPR/Cas technique and one using • Amount of tolerated mismatches at the potential of- the TALENs technique. No study analyzed of-target target sites. efects using unbiased detection methods in ZFN, ODM, • Quality of available biased detection methods. MN and BE studies. • CRISPR variants (e.g. Nickase). • Chosen sgRNA. Knowledge clusters • Methodology of SDN delivery. Te analysis of of-target efects in CRISPR/Cas studies using biased detection methods represents a knowledge cluster. Tis cluster is amenable for a critical appraisal Limitations of the systematic map serving the interest of researchers and decision-makers. References were only searched in German and English Te following parameters could afect the occurrence of language. Publications in an Asian language, in which of-targets and are worth to be analyzed in-depth: only the abstract was available in English, were also Modrzejewski et al. Environ Evid (2019) 8:27 Page 27 of 33

Fig. 11 Overview of the used approaches to assess of-target efects in TALENs studies. All numbers represent the amount of studies conducted for diferent approaches

identifed but could not be included in this map for the It has been demonstrated that the individual stud- extraction of detailed data. Since most of the studies were ies addressing of-target efects difer widely in design found in scientifc journals, a bias in the pool of articles and implementation. Terefore, no reliable conclusions found is that publications from countries that probably about the occurrence of of-target efects can be drawn use genome editing but do not publish in either English based on the results of this map. A critical appraisal of or German language are not represented. For example, the individual studies in form of a systematic review is it can be assumed that in South American countries, recommended. considerably more research is done than the identifed literature suggests. Identifying grey literature as well as Conclusions manually identifying scientifc journals publishing in the Implications for policy/management local language(s) was also not possible due to language Tis systematic map identifed substantial bodies of evi- barriers. dence regarding the applications of diferent genome- Additionally, the full text of 104 articles, that have been editing techniques in plants as well as the occurrence of rated as relevant on title/abstract level, were un-retriev- of-target efects. Until May 2018, almost 100 market- able and were therefore not included in the systematic oriented applications were identifed including improved map (see Additional fle 3). food and feed quality, yield improvement, altered growth Modrzejewski et al. Environ Evid (2019) 8:27 Page 28 of 33

the chosen sgRNA. A critical appraisal in the course of a systematic review could help to identify parameters in order to further reduce the occurrence of of-target efects. Te identifed knowledge cluster for the detec- tion of of-target efects in CRISPR-studies using biased detection methods would be suitable to address this. Te results of this map further identifed a knowledge gap regarding the analysis of of-target efects using unbi- ased detection tools. To increase this data basis and to evaluate whether an unbiased of-target analysis has an added value compared to biased of target analysis, more studies should assess this aspect, in future.

Additional fles

Additional fle 1. Detailed description of the genome-editing techniques. Additional fle 2. List of peer-reviewed and grey literature sources searched. Additional fle 3. List of un-retrievable articles. Additional fle 4. List of articles excluded at full text assessment with the reason for exclusion. Additional fle 5. List of relevant studies and the systematic map data- base to answer secondary question 1. Fig. 12 Overview of the used approaches to assess of-target Additional fle 6. List of relevant studies and the systematic map data- efects in BE studies. All numbers represent the amount of studies base to answer secondary question 2. conducted for diferent approaches Additional fle 7. ROSES Reporting Form.

Acknowledgements characteristics, resistance against biotic and abiotic The authors wish to thank Maren Fischer for carefully reading the manuscript. stress, herbicide tolerance and industrial utilization. Te wide range of diferent applications addressing all parts in Authors’ contributions The review process was coordinated by DM. DM, FH, TS and DK conducted the the agricultural value chain and the application in many screening of articles and data extraction. DM drafted the report. FH, TS, CK and diferent plant species indicates that genome-editing RW assisted in editing and revising the draft. All authors read and approved became a promising tool to breed varieties that are better the fnal manuscript. adapted to the needs of agriculture and to enable a valu- Funding able contribution to food security and the environment. This systematic map is part of the ELSA-GEA project funded by the Federal A decisive factor that impacts the use of genome- Ministry of Education and Research (BMBF) (01GP1613B). editing is the acceptance by consumers and retailers for Availability of data and materials products derived from innovative plant breeding meth- The datasets generated and/or analyzed during the current study are available ods. A prominent point of criticism in this context is the at the ELSA-GEA homepage https​://www.dialo​g-gea.de/de/servi​ce/repos​itori​ um. occurrence of of-target efects. Since plant breeding is the contextual background, it is worth to compare the Ethics approval and consent to participate occurrence of of-target efects in the context of naturally Not applicable. occurring mutations and routinely used breeding tech- Consent for publication niques like regular crossing or undirected mutagenesis Not applicable. using chemical mutagens or irradiation [10]. Competing interests The reviewers Frank Hartung (FH) and Thorben Sprink (TS) are authors of a few Implication for research research studies included in the review process. However, as none of these Results of this systematic map determined that difer- studies comprise primary data, their articles were excluded at title/abstract level. ent approaches were used to analyze of-target efects depending on the plant species and with regard to the Received: 28 March 2019 Accepted: 27 June 2019 CRISPR-variant, the prediction tools and detection methods used, the amount of tolerated mismatches and Modrzejewski et al. Environ Evid (2019) 8:27 Page 29 of 33

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